Pathways of TRH degradation in rat brain

We have investigated the TRH degradative enzymes in brain by examining the pattern of metabolites formed . The homogenate and three subcellular fractions of rat brain were separately incubated for 1, 5, and 15 minutes with ³H-TRH (pyro-glu-his-³H-proNH₂) at 37°C. TRH and its metabolites were separated on silica gel thin-layer chromatography plates. The crude homogenate and subcellular fractions each produced a characteristic pattern of metabolism. The homogenate metabolized TRH to TRH-OH, proline, and prolineamide. With the P₁ fraction, prolineamide and proline were the major metabolites. In both the homogenate and P₁ fraction incubations, histidyl-prolineamide appeared as a minor component. The P₂ fraction produced prolineamide and histidyl-prolineamide as the major metabolites while the cytosol metabolized TRH primarily to TRH-OH. Proline was formed during incubation with both cytosol and P₂ fractions.The TRH deamidase is found in the soluble fraction of brain tissue homogenate while the pyroglutamate aminopeptidase and the prolineamide cleaving enzyme are associated with particulate fractions. Histidyl-prolineamide is further degraded in the homogenate and P₁ fractions by a secondary metabolic pathway. Proline salvaging enzymes are present in all subcellular fractions of rat brain.